The purpose of this invention is to expand the number of incitants that can be utilized in allergy testing to determine which antigens or combinations of antigens are causing patient symptoms. As ozone levels and industrial pollution increase, the population at large is becoming more susceptible to adverse reactions from many sources. These include additives in food, food articles, pollens, molds, terpenes, chemicals, weeds, trees, and grasses.
Outdoor air pollution has long been thought to enhance, or at times even cause disease processes. Hippocrates mentions it in his work Air, Waters, and Places. Increased industrialization and mechanization that began around 1950 accelerated the growth of anthropogenic (manmade) pollution. According to a recent estimate, there are presently over 70,000 chemicals now in commercial production and another 700-3,000 new chemicals are being introduced every year. The U.S. Environmental Protection Agency lists 188 hazardous air pollutants either known or suspected of causing cancer, serious human health effects or ecosystem damage.
As the human immune system is taxed to detoxify from exposure to pollution, the body has become sensitized to a wider range of chemicals and natural pollutants. As a result, individuals are experiencing a wide range of both chronic and acute symptoms related to these exposures.
Presently, allergy testing commonly takes the form of intracutaneous skin tests, patch tests and blood testing such as the Radioallergosorbent Test (RAST). Skin testing comprises introducing allergens to a portion of the patients"" dermis and then measuring the patients"" response to the allergen. Various methods are known and used to measure the patient""s response. For example, the size and color of any induced weal can be measured. U.S. Pat. No. 5,413,113 to Milne, incorporated herein by reference, describes methods of measuring galvanometric skin response to an allergen.
The skin patch is a similar method of testing for allergies and irritants. A suspected allergen or irritant is applied to normal skin under occlusion for a period of time, this application being in a controlled manner and with a suitable formulation and concentration of the test substance. Allergic eczema or irritative eczema reactions indicate a patient""s sensitivity to the suspect substance. Usually the test substance is applied in a dispersion of petrolatum or aqueous solution. Means of holding the test substance in contact with the patient""s dermis include the use of a foil cup or polymeric film as disclosed in U.S. Pat. No. 4,836,217 to Fischer, et al. and incorporated herein by reference.
Over 2000 substances are known as common test substances in the testing for allergens. In 1984 the American Academy of Dermatology established a list of test substances. The earlier list included: Benzocain (5% petrolatum), imidazolidinyl urea (2% aq), Thiuram mix (1% petrolatum), cinnamic alcohol (5% petrolatum) dibucaine (1% petrolatum), mercaptobenzothiazole (1% petrolatum), neomycin sulfate (20% petrolatum), p-phenylenedimine (1% petrolatum), tetracaine (1% petrolatum), p-tert.butylphenol formaldehyde resin (1% petrolatum), Thiomersal (0.1% petrolatum), formaldehyde (2% aq.), hydroxycitronellal (4% petrolatum), Carba mix (3% petrolatum), cinnamic aldehyde (2% petrolatum), rosin (colophony) (20% petrolatum), PPD mix (black rubber mix) (0.6% petrolatum), wool (lanolin) alcohols (30% petrolatum), cyclomethycaine (1% petrolatum), eugenol (4% petrolatum), Quaternium-15 (2% petrolatum), isoeugenol (4% petrolatum), mercapto mix (1% petrolatum), epoxy resin (1% petrolatum), potassium dichromate (0.5% petrolatum), caine mix less benzocaine (3% petrolatum), ethylenediamine dihydrochloride (1% petrolatum), benzoyl peroxide (1% petrolatum), balsam of Peru (25% petrolatum), Quaternium-15 (2% aq.), oak moss (abs. mousse de chene) (5% petrolatum), nickel sulfate (2.5% petrolatum). This list has subsequently been revised by the North American Contact Dermatits Group. As of 1999 this list has expanded to 50 substances with an additional 15 to be added by the year 2001. Similar lists of testing substances are available for Europe and Japan.
In skin tests, a number of apparatus have been described that introduce the allergen test substance into contact with a patient""s dermis. For example, U.S. Pat. No. 3,289,670, incorporated herein by reference, discloses an apparatus that produces multiple cutaneous sites by abrading the skin and applying test substances. Intracutaneous injection of multiple test substances is disclosed in U.S. Pat. No. 4,270,548 to Brennan, incorporated herein by reference. In contrast to the test substances used in patch tests, intracutaneous allergy testing often concentrates on airborne allergies such as tree allergens, mold allergens, grass allergens, ragweed allergens, weed allergens, dust, epidermals (dander, animal hair, feathers, etc) and foods.
The intracutaneous extracts used for food testing include testing for the food groups including: whole cow""s milk, whole egg, legumes, chocolate, grains (wheat, rye, barley, corn), citrus fruits (orange, grapefruit, lemon), potato family (potato, tomato, green pepper), seafood (with fish family) and cucumber (cucumber, cantaloupe, watermelon).
Intracutaneous test substances are commonly provided in either an aqueous form or in a glycerin-saline base. The aqueous form comprises a preservative in addition to the allergen and normal saline solution. For example, the 0.5% methyl paraben and 0.05% propylparaben can be added to the aqueous forms. Other aqueous preservatives include phenol and albumin. The glycerin-saline base is comprised of 50% glycerin so that additional preservative is not required. The preservative is added to increase the shelf-life of the test substance. This allows standardized test substances to be produced in a controlled environment and distributed to clinicians in an economical fashion.
However, it has been noted that some patients are sensitive to the preservatives used in the test substance preparations. The American Journal of Contact Dermatology reports of cases of allergic contact dermatitis from patient skin contact with paraben preservatives. Similarly, other patients may be allergic to glycerin, phenol or albumin. When such patients receive a series of allergy tests, they show xe2x80x9cfalse positivesxe2x80x9d caused by their allergic reaction to preservative and not the named test substance.
Typical preparation of allergen extracts for intradermal use begins with a powder or ground solid form of the allergen. This powder is then dissolved or dispersed into an aqueous or normal saline solution and then filtered. Due to the nature of these powders, they often comprise the named allergen along with other foreign matter. This foreign matter is deemed to be a contaminant that lowers the specificity of the allergen extract. Patients may be allergic to the foreign matter contaminants while not being allergic to the named allergen, thus showing a xe2x80x9cfalse positive.xe2x80x9d
Of the different forms of contaminants, mold, bacteria and endotoxin can be present. Special efforts must be made to insure that these forms are reduced to acceptable levels.
An additional consideration in the preparation of allergen extracts is the ability to prepare extracts of consistent biological activity. Medical clinicians rely upon this consistency in order to obtain reproducible, valid test results. Allergen extracts that are excessively active may cause allergic shock dangers to patients while extracts that are excessively inactive will not properly identify allergic responses.
As a result, there is a need for prepared allergen extracts with adequate shelf life that comprise the named test substance without added preservatives and that further have a known biological activity.
It is an object of the present invention to provide allergen testing preparations and testing protocols that are more sensitive and accurate than existing methods.
It is a further object of the present invention to provide allergen testing preparations of known shelf life and standardized biological activity that do not contain preservative agents.
It is yet another object of the present invention to provide methods of extending the shelf life of such prepared allergen testing preparations.
In accordance with one embodiment of the present invention, a method is provided for preparing preservative-free allergen test solutions in a prepared sterile environment. The method comprises preparing a sterile environment by utilizing a disinfectant wipe and a sterile barrier field. An antigen is added to a diluent to form a solution of the antigen by dispersing or suspending the antigen in the diluent. Optionally, normal saline is used as the diluent. The initial solution is maintained at a temperature selected to impede bacterial and mold growth for a period of at least 12 hours. In one embodiment, the initial solution is maintained at a temperature from 33xc2x0 F. to 38xc2x0 F. The initial solution is passed through a screen and first filter to become the first filtrate, wherein the screen is USP type gauze and the first filter has a pore size of 12 microns or less. The first filtrate is passed through a vacuum filter having a pore size of 2 microns. The resultant filtrate is passed through a vacuum filter having a pore size of 1.5 microns to become the second filtrate. The second filtrate is passed through a vacuum filter having a pore size of 0.22 micron. The resultant filtrate is subsequently passed through a filter having a pore size of 0.1 micron into a sterile product container. In accordance with one embodiment, the allergen test solutions are prepared to a consistent protein nitrogen concentration.
The process of the present invention allows extension of the shelf-life of the allergen test solution by storing the solution at a temperature below 5xc2x0 F. Another method for extending the shelf life of the allergen of the present invention is by lyophilizing the allergen test solution.
In another embodiment, the present invention provides a method of preparing preservative-free allergen test solutions in a prepared sterile environment. The method comprises preparing a sterile environment by utilizing a disinfectant wipe and a sterile barrier field and adding an adding an antigen to a diluent solution, such as normal saline. The antigen is dispersed or dissolved in the diluent solution to form an initial supension. The initial initial solution is maintained at a temperature selected to impede bacterial and mold growth for a period of at least 12 hours. The initial solution is subjected to centrifuging for a minimum of 30 minutes, transferring a minimum of 98% by volume of the centrifuged solution to a sterile container. Diluent is added to increase the volume to 100% of the initial solution volume. The resultant solution is passed through a vacuum filter having a pore size of 0.22 micron with the resultant filtrate passed through a filter having a pore size of 0.1 micron into a sterile product container.
This invention relates to the method of preparing allergen testing solutions without the use of preservatives and of known biological activity, enhanced purity and known shelf-life. The invention further comprises the preferred method of extending shelf-life for these test substances.
In accordance with one embodiment of the present invention, a method is provided for preparing preservative-free allergen test solutions in a prepared sterile environment. The method comprises preparing a sterile environment by utilizing a disinfectant wipe and a sterile barrier field. An antigen is added to a diluent to form a solution of the antigen by dispersing or suspending the antigen in the diluent. Optionally, normal saline is used as the diluent. The initial solution is maintained at a temperature selected to impede bacterial and mold growth for a period of at least 12 hours. In one embodiment, the initial solution is maintained at a temperature from 33xc2x0 F. to 38xc2x0 F. The initial solution is passed through a screen and first filter to become the first filtrate, wherein the screen is USP type gauze and the first filter has a pore size of 12 microns or less. The first filtrate is passed through a vacuum filter having a pore size of 2 microns. The resultant filtrate is passed through a vacuum filter having a pore size of 1.5 microns to become the second filtrate. The second filtrate is passed through a vacuum filter having a pore size of 0.22 micron. The resultant filtrate is subsequently passed through a filter having a pore size of 0.1 micron into a sterile product container. In accordance with one embodiment, the allergen test solutions are prepared to a consistent protein nitrogen concentration.
The process of the present invention allows extension of the shelf-life of the allergen test solution by storing the solution at a temperature below 5xc2x0 F. Another method for extending the shelf life of the allergen of the present invention is by lyophilizing the allergen test solution.
In another embodiment, the present invention provides a method of preparing preservative-free allergen test solutions in a prepared sterile environment. The method comprises preparing a sterile environment by utilizing a disinfectant wipe and a sterile barrier field and adding an adding an antigen to a diluent solution, such as normal saline. The antigen is dispersed or dissolved in the diluent solution to form an initial supension. The initial initial solution is maintained at a temperature selected to impede bacterial and mold growth for a period of at least 12 hours. The initial solution is subjected to centrifuging for a minimum of 30 minutes, transferring a minimum of 98% by volume of the centrifuged solution to a sterile container. Diluent is added to increase the volume to 100% of the initial solution volume. The resultant solution is passed through a vacuum filter having a pore size of 0.22 micron with the resultant filtrate passed through a filter having a pore size of 0.1 micron into a sterile product container.
The allergen testing solutions are presented in Table 1 and Table 2. Table 1 indicates testing solutions comprising multiple related allergens. Table 2 indicates testing solutions comprising specific allergens.
Preservative-free allergen test solutions are prepared by following the triple filtration process as described below. These solutions are tested for shelf-life by determining antigenicity of stored solutions against solutions less than 48 hours old. Three sets of prepared solutions are used for this shelf-life study. The first set is stored at a temperature of from xe2x88x9210 to 10xc2x0 F., in substantially a solid state. The second set is stored at a temperature of from 39 to 45xc2x0 F. after first being exposed to ultraviolet light sterilization.
The third set of test solutions are dehydrated by controlled temperature dehydration, utilizing a Lyopholizer apparatus. Following the waiting of various shelf-life periods, these test solutions are reconstituted with intravenous injection grade purified water and then tested for antigenicity.
As used below the term xe2x80x9cidentifying protocolxe2x80x9d shall refer to established test methods for verification of strength and composition of the various allergens. For example, allergens that are proteinaceous in nature commonly use protein nitrogen units (PNU) and total nitrogen content as elements of the identifying protocol. Other methodology used in identifying protocols includes petri dish assay and spectrophotometric analysis.
Consistency and standardization are important to executing a quality step-by-step antigen filtration process. Using known methods of scientific research that are highly regarded among the medical community is the best method in proving the validity of a new idea. A difference of less than 10% of control parameter is the goal for each antigen. The following examples indicate methods important to the production of allergen test solutions.